T_reg cells communicate with allogeneic DC directly via cell-to-cell contact and GJIC dependent manner.

Bone marrow derived dendritic cells (BMDC from BALB/c mice, 3×10⁴ per well) were cocultured with B6.SJL CD45.1⁺ T cells and C57BL/6 CD45.1⁻ calcein-labelled pre T_reg cells for 20 h in a 1∶1∶3 ratio. (A) Transfer of green fluorescent calcein after 20 h was analyzed by gating on CD45.1⁻CD11c⁺ DC, CD45.1⁻CD11c⁻ T_reg cells and CD45.1⁺CD11c⁻ T cells, respectively. (B) Differences of median fluorescence intensity (MFI) among BMDC, CD4⁺ and CD8⁺ T cells was assessed. Where indicated T_reg cells were preincubated with the gap junction inhibitor GAP27 (300 ng/ml). CFDA-SE (1 µM) labelled T_reg cells were used as a control. (C) BALB/c BMDC were left untreated or cocultured with C57BL/6 pre T_reg cells in an 1∶1 ratio in presence of soluble anti-CD3-mAb (3 µg/ml). Where indicated GAP27 (300 ng/ml) or rolipram (300 nM) were added. After 4 h suppressed DC (DC _sup) were purified by CD11c specific MACS. Intracellular cAMP concentration was assessed by a specific ELISA after lysis of the cells. (D) Changes in cAMP levels after a 4 h coculture with BALB/c BMDC were mesured by flow cytometry in C57BL/6 preT_reg. (*) indicates significant differences by Mann-Whitney U-test. The data shown are representative for 2 independent experiments in duplicate or triplicate wells.